Familial defective apolipoprotein (apo) B100 (FDB) is a genetic abnormality of lipid metabolism that causes moderate to severe hypercholesterolemia. A mutation in apo-B100 severely diminishes the ability of low density lipoproteins (LDL) to bind to the LDL receptor. Therefore, LDL accumulate in the plasma because their efficient receptor- mediated catabolism is disrupted. There is a strong association of the mutation (Arg3500 -> Gln) in apo-B100 with FDB. This mutation is widespread in the United States and Europe and, in some populations, this genetic abnormality is the first or second most prevalent genetic cause of hypercholesterolemia and premature atherosclerosis. One of the main objectives of this application is to prove that the Arg3500 -> Gln mutation causes FDB and to understand how the mutation disrupts LDL receptor binding. A full-length apo-B minigene with and without the 3500 mutation will be expressed in hepatic cells in culture and in transgenic mice. The "LDL" or LDL-like particles will be isolated from cell culture media and from the plasma of transgenic mice, characterized, and tested for LDL receptor binding. We will test the hypothesis that the 3500 mutation is in a region of apo-B that does not interact directly with the LDL receptor, but modulates or alters the conformation of the binding site. As part of our investigations on FDB, new mutations in apo-B100 that cause defective LDL binding will be sought by examining the ability of LDL isolated from hypercholesterolemic subjects to bind to LDL receptors. We will also take advantage of the FDB mutation to learn more about normal VLDL, IDL, LDL and Lp(a) metabolism. The second main objective is to define the receptor binding domain of apo-B100 and determine which amino acids are crucial for apo-B100 binding to the LDL receptor. Part of this objective is to determine the affinity of LDL from eight vertebrate species for LDL receptors on normal human fibroblasts. Comparison of the putative receptor binding domain sequence with the receptor binding ability of the LDL should reveal highly conserved regions critical for this function which will then be targets for mutagenesis. Site-directed mutagenesis will alter or delete the site and residues of apo-B100 we believe are critical for LDL binding. The mutated apo-B100 minigene will be expressed in cultured cells and transgenic mice, characterized thoroughly, and tested for receptor binding. The elucidation of the receptor binding site of apo-B100 and the understanding of how certain mutations of apo-B100 disrupt LDL binding will further our growing knowledge about the interaction of apo- B100 with the LDL receptor and the molecular basis of genetic abnormalities that cause hypercholesterolemia and premature atherosclerosis.